Uveitis In Rats Research Articles

Antidiabetic Drug Metformin Suppresses Endotoxin-Induced Uveitis in Rats

To investigate the therapeutic effects of metformin, a commonly used antidiabetic drug, in preventing endotoxin-induced uveitis (EIU) in rats. EIU in Lewis rats was developed by subcutaneous injection of lipopolysaccharide (LPS; 150 μg). Metformin (300 mg/kg body weight, intraperitoneally) or its carrier was injected either 12 hours before or 2 hours after LPS induction. Three and 24 hours after EIU, eyes were enucleated and aqueous humor (AqH) was collected. The MILLIPLEX-MAG Rat cytokine-chemokine magnetic bead array was used to determine inflammatory cytokines. The expression of Cox-2, phosphorylation of AMPK, and NF-κB (p65) were determined immunohistochemically. Primary human nonpigmented ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of metformin. Compared with controls, the EIU rat AqH had significantly increased number of infiltrating cells and increased levels of various cytokines and chemokines (TNF-α, MCP-1, IL-1β, MIP-1α, IL-6, Leptin, and IL-18) and metformin significantly prevented the increase. Metformin also prevented the expression of Cox-2 and phosphorylation of p65, and increased the activation of AMPK in the ciliary bodies and retinal tissues. Moreover, metformin prevented the expression of Cox-2, iNOS, and activation of NF-kB in the HNPECs and decreased the levels of NO and PGE2 in cell culture media. Our results for the first time demonstrate a novel role of the antidiabetic drug, metformin, in suppressing uveitis in rats and suggest that this drug could be developed to prevent uveitis complications.

Traditional Japanese Herbal (Kampo) Medicines and Treatment of Ocular Diseases: A Review

Herbal medicines have been used clinically in Eastern Asia, and traditional Japanese herbal (Kampo) formulas are approved as ethical drugs. The Kampo formulas are mixtures of the crude extracts of several herbs, each of which contains multiple components. Numerous investigators have reported that some herbal medicines are efficacious for treating several human diseases. We reviewed the literature on traditional herbal medicines and treatment of ocular diseases. Oral Orengedoku-to and Kakkon-to inhibit postoperative uveitis in humans. Oral Goshajinki-gan improved ocular surface disorders in patients with type 1 diabetes mellitus. Oral Hachimijio-gan increased retinal blood flow. Keishi-bukuryo-gan Sho might be associated with vitreoretinopathy in patients with type 2 diabetes mellitus. Oral Hachimijio-gan and Goshajinki-gan delayed lens opacification in rats and mice. Oral Sairei-to, Orengedoku-to, Senkanmeimoku-to, Scutellariae radix extract, Gardeniae fructus extract, topical Liguisticum wallichii rhizoma extract, and intravenous injection of tetramethylpyrazine, baicalin, baicalein, wogonin, and crocetin inhibited some forms of experimental uveitis in rabbits. Topical glycyrrhizinate improved allergic conjunctivitis in humans and rats. Oral crocetin improved eyestrain in humans. Oral berberine diminished experimental uveitis in rats. Baicalein, wogonin, berberine, and berberrubine inhibited in vitro expression of several cytokines in cultured retinal pigment epithelial cells. Some Kampo formulas are efficacious for treating several ocular diseases in humans and animals. Some herbal extracts and their components inhibit some forms of experimental uveitis.

Qingkailing Injection Alleviates Experimental Autoimmune Uveitis in Rats <i>via</i> Inhibiting Th1 and Th17 Effector Cells

Qingkailing injection is a well-known composite formula of traditional Chinese medicine and is commonly used in clinical practice, which could offer immunomodulatory effect from our clinical experience on uveitis treatment by Qingkailing. We did the experiment in order to investigate the curative effect and mechanism of Qingkailing injection to rat experimental autoimmune uveitis (EAU). EAU was induced in Lewis rats by immunization IRBP1177-1191 in complete Freund's adjuvant in multi-point. We found that Qingkailing injection can alleviate autoimmune uveitis in rats, inhibit the differentiation toward Th1 and Th17 effector cells and the relevant cytokines secretion. The therapeutic effect may also be regulated through increased secretion of interleukin (IL)-10.

Effects of Instillation of Eyedrops Containing Disulfiram and Hydroxypropyl-β-Cyclodextrin Inclusion Complex on Endotoxin-Induced Uveitis in Rats

Objective: To investigate the anti-inflammatory effects of the instillation of disulfirum (DSF) eyedrops that enhance solubility using 2-hydroxypropyl-β-cyclodextrin (HPβCD) and hydroxypropylmethylcellulose (HPMC) on endotoxin-induced uveitis (EIU) in rats and mechanisms related to ocular inflammation.Methods: EIU was induced in male Lewis rats by subcutaneous injection of 200 μg lipopolysaccharide (LPS). DSF (0.125%, 0.25% and 0.5%) or commercially available 0.05% dexamethasone (Dexa) was topically applied to both eyes of rats 1 hour before, immediately after, and 1 and 2 hours after injection of LPS. The aqueous humor (AqH) was collected 24 hours after LPS injection, and the number of infiltrating cells, protein concentration, and levels of tumor necrosis factor-α (TNF-α), nitric oxide (NO) and prostaglandin E2 (PGE2) were determined. Immunohistochemical analysis of the iris ciliary body (ICB) cells was performed to determine the expression of activated nuclear factor κB (NF-κB), inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).Results: The topical administration with DSF suppressed, in a dose-dependent manner, the number of inflammatory cells, the protein concentration, and the levels of NO, TNF-α and PGE2 in the AqH and improved the histologic status of the ocular tissue. The anti-inflammatory potency of 0.5% DSF treatment was as strong as that of 0.05% Dexa. Topical DSF treatment also suppressed the activated NF-κB 3 hours after LPS injection, and iNOS and COX-2 expression in the ICB 24 hours after LPS injection.Conclusions: The present results demonstrate that the topical instillation of DSF eyedrops suppresses the inflammation in EIU, suggesting a possible novel approach for the treatment of ocular inflammation.

Amelioration of experimental autoimmune uveoretinitis by aldose reductase inhibition in Lewis rats.

Recently, the authors showed that the inhibition of aldose reductase (AR) prevents bacterial endotoxin-induced uveitis in rats. They have now investigated the efficacy of AR inhibitors in the prevention of experimental autoimmune-induced uveitis (EAU) in rats. Lewis rats were immunized with bovine interphotoreceptor retinoid-binding peptide (IRBP) to develop EAU. Two or 8 days after immunization, the rats started receiving the AR inhibitor fidarestat (7 mg/kg/d; intraperitoneally). They were killed when the disease was at its peak; aqueous humor (AqH) was collected from one eye, and the other eye of each rat was used for histologic studies. The protein concentration and the levels of inflammatory markers were determined in AqH. Immunohistochemical analysis of eye sections was performed to determine the expression of inflammatory markers. The effect of AR inhibition on immune response was investigated in isolated T lymphocytes. Immunization of rats by IRBP peptide resulted in a significant infiltration of leukocytes in the posterior and the anterior chambers of the eye. Further, EAU caused an increase in the concentration of proteins, inflammatory cytokines, and chemokines in AqH, and the expression of inflammatory markers such as inducible-nitric oxide synthase and cycloxygenase-2 in the rat eye ciliary bodies and retina. Treatment with fidarestat significantly prevented the EAU-induced ocular inflammatory changes. AR inhibition also prevented the proliferation of spleen-derived T cells isolated from EAU rats in response to the IRBP antigen. These results suggest that AR could be a novel mediator of bovine IRBP-induced uveitis in rats.

Inhibitory Effect of Aminoimidazole Carboxamide Ribonucleotide (AICAR) on Endotoxin-Induced Uveitis in Rats

PURPOSE. To investigate the anti-inflammatory effect of aminoimidazole carboxamide ribonucleotide (AICAR), an analog of adenosine monophosphate (AMP), in endotoxin-induced uveitis (EIU). METHODS. EIU was induced by subcutaneous injection of lipopolysaccharide (LPS) (200 μg) in Lewis rats. AICAR (50 mg/kg, intraperitoneally) was given 6 hours prior and at the same time as LPS injection. Clinical uveitis scores, number of anterior chamber (AC) infiltrating cells, anterior chamber protein concentration, retinal vessel leukocyte adhesion, and protein leakage were measured 24 hours later. Protein levels of C-C chemokine ligand-2 (CCL-2)/monocyte chemotactic protein-1 (MCP-1), tumor necrosis factor-α (TNF-α) and intercellular adhesion molecule-1 (ICAM-1) in aqueous humor and retina and nuclear translocation of nuclear factor-κB (NF-κB) in the retina were determined by enzyme-linked immunosorbent assay (ELISA). Both mRNA and protein levels of CD14 in peripheral blood mononuclear cells were also measured. RESULTS. AICAR treatment significantly reduced EIU clinical severity as well as inflammatory cell infiltration and protein concentration in aqueous humor. Similarly, the number of retinal vessel-adherent leukocytes and protein leakage were decreased by AICAR treatment. Protein levels of TNF-α, CCL-2/MCP-1, and ICAM-1 in aqueous humor and CCL-2/MCP-1 and ICAM-1 levels in retina were suppressed with AICAR treatment. AICAR also reduced NF-κB translocation and CD14 expression. CONCLUSIONS. AICAR reduces systemic LPS susceptibility and attenuates intraocular inflammation in a rat EIU model by limiting infiltration of leukocytes, suppressing inflammatory mediators, and inhibiting the NF-κB pathway.

Preventive Effects of Ethyl Pyruvate on Endotoxin-Induced Uveitis in Rats

Recent studies indicate that ethyl pyruvate (EP) exerts anti-inflammatory properties; however, the effect of EP on ocular inflammation is not known. The efficacy of EP in endotoxin-induced uveitis (EIU) in rats was investigated. EIU in Lewis rats was developed by the subcutaneous injection of lipopolysaccharide (LPS; 150 μg). EP (30 mg/kg body weight) or its carrier was injected intraperitoneally 1 hour before or 2 hours after lipopolysaccharide injection. Animals were killed after 3 and 24 hours followed by enucleation of eyes and collection of the aqueous humor (AqH). The number of infiltrating cells and levels of proteins in the AqH were determined. The rat cytokine/chemokine multiplex method was used to determine level of cytokines and chemokines in the AqH. TNF-α and phospho-nuclear factor kappa B (NF-κB) expression in ocular tissues were determined immunohistochemically. Human primary nonpigmented ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of EP on lipopolysaccharide-induced inflammatory response. Compared to controls, AqH from the EIU rat eyes had a significantly higher number of infiltrating cells, total protein, and inflammatory cytokines/chemokines, and the treatment of EP prevented EIU-induced increases. In addition, EP also prevented the expression of TNF-α and activation of NF-κB in the ciliary bodies and retina of the eye. Moreover, in HNPECs, EP inhibited lipopolysaccharide-induced activation of NF-κB and expression of Cox-2, inducible nitric oxide synthase, and TNF-α. Our results indicate that EP prevents ocular inflammation in EIU, suggesting that the supplementation of EP could be a novel approach for the treatment of ocular inflammation, specifically uveitis.

Effect of Artesunate on Endotoxin-Induced Uveitis in Rats

The purpose of this study was to explore the effect of artesunate on endotoxin-induced uveitis (EIU) in rats. EIU was induced in male Long-Evans rats by footpad injection of lipopolysaccharide (LPS 200 μg in 0.1 mL PBS; LPS-only group [LG]). PBS (0.1 mL), prednisolone (10 mg/kg in 0.1 mL PBS; prednisolone group [PG]), and artesunate of three concentrations (1, 10, 100 mg/kg; artesunate groups A, B, C [AGA, AGB, AGC]; all in 0.1 mL of PBS) were injected intravenously, respectively, 1 hour after LPS injection. Twenty-four hours after LPS injection, infiltrating cells and protein concentration in the aqueous humor, as well as tumor necrosis factor (TNF)-α, nitric oxide (NO), prostaglandin E2 (PGE2), and monocyte chemoattractant protein (MCP)-1, were measured. Rats in AGA did not have significant difference in infiltrating cell number (61.6 × 10(5) cells/mL; range, 20.0-147.8 cells/mL; P = 0.287), protein concentration (48.0 mg/mL; range, 18.0-101.8 mg/mL; P = 0.349), NO concentration (86.9 μM/L; range, 30.5-192.5 μM/L; P = 0.363), but had significant difference in TNF-α (1144.3 pg/mL; range, 460.0-1840.0 pg/mL; P = 0.038), PGE2 (12.8 ng/mL; range, 4.8-27.2 ng/mL; P = 0.005), and MCP-1 (6136 pg/mL; range, 20.0-147.8 pg/mL; P = 0.009) with those of LG. Artesunate at 10 mg/kg and 100 mg/kg significantly suppressed the infiltrating cells, protein concentration, TNF-α, NO, PGE2, and MCP-1 in the aqueous humor induced by LPS (P = 0.011-0.000). Dose-dependent decreases of infiltrating cells, protein concentration, TNF-α, PGE2, NO, and MCP-1 in the aqueous humor by artesunate treatment after LPS injection indicate that artesunate can suppress the inflammation of EIU by inhibiting the production of inflammatory mediators.

Anti-Inflammatory Potency of Oral Disulfiram Compared with Dexamethasone on Endotoxin-Induced Uveitis in Rats

To investigate potency of oral disulfiram (DSF) compared with that of dexamethasone (Dexa), on endotoxin-induced uveitis (EIU) in rats. The oral administration with 750 mg/kg DSF suppressed the number of inflammatory cells, protein concentration, and levels of tumor necrosis factor (TNF)-α, Nitric oxide (NO) and prostaglandin (PG) E2 in the aqueous humor and improved the histiologic status of the ocular tissue at 24 hr after lipopolysaccharide (LPS) injection. The anti-inflammatory potency of DSF oral administration was as strong as that observed with 0.5 mg/kg Dexa in the present study. The results suggest that DSF might pave the way for a novel therapeutic agent for the management of uveitis.

Transplantation of bone marrow mesenchymal stem cells inhibits rat experimental autoimmune uveitis

Objective To investigate the impact of bone marrow mesenchymal stem cell transplantation on a rat model of experimental autoimmune uveitis (EAU) and analyze its immune regulatory mechanisms in vivo. Methods Eighteen Lewis rats were randomly divided into three groups: model control group, intervention group and normal control group, six animals in each group. Human retinal S-antigen peptide (HS-AgP35, 1 mg/ml) was mixed and emulsified with complete Freund's adjuvant and injected into hind foot pad of rats on the first and eighth day to establish the animal model of EAU. For bone marrow mesenchymal stem cell transplantation, 1 ml of cell suspension (2 × 106 cells/ml) was injected into tail vein of the intervention group rats on the first day when the emulsified S-antigen was injected. EAU manifestation, pathological change and IFN-γ level were evaluated and compared among those three groups after two weeks. Results No abnormal signs were found in the eyes of rats in normal control group. The manifestation grading of the intervention group (two rats at grade 0, three rats at grade 0.5, one rat at grade one) was significantly different from the model control group (one rat at grade one, one rat at grade two, three rats at grade three, one rat at grade four) (P=0. 015). The retina of rats in normal control group was ordinary under light microscope. The histopathological grading of the intervention group (one rat at grade 0, four rats at grade 0.5, one rat at grade one) and the model control group (four rats at grade three, two rats at grade four) was also statistically different (P＜0. 01). Furthermore, the IFN-γ level in peripheral blood of the intervention group rats declined significantly compared to the model control group (t=9. 057 4, P=0. 01). Conclusions Bone marrow mesenchymal stem cells can inhibit EAU significantly,possibly by lowering the level of IFN-γ, thereby reduce the severity of uveitis and improve the condition of uveitis in rats. Key words: Uveitis/therapy; Myeloid progenitor cells/transplantation; Mesenchymal stem cells; Animal experimentation

Inhibitory Effects of Oral Disulfiram on Endotoxin-Induced Uveitis in Rats

Purpose: Disulfiram (DSF) exhibits a wide variety of biological activities, including an anti-inflammatory action, on which we focused our attention. The aim of the present study was to investigate the effect of oral DSF on endotoxin-induced uveitis (EIU) in rats.Methods: We investigated its effect upon cellular infiltration and protein leakage, as well as on the concentration of tumor necrosis factor-α (TNF-α), nitric oxide (NO), and prostaglandin E2 (PGE2) in the anterior chamber. Some eyes were enucleated for histologic examination and immunohistochemical analysis. EIU was induced in male Lewis rats by a footpad injection of lipopolysaccharide (LPS). One hour before the LPS injection, either 250, 500, or 750 mg/kg DSF was administered orally. Twenty-four hours later, the aqueous humor was collected from both eyes, and the number of infiltrating cells and protein concentration in the aqueous humor were determined. Levels of TNF-α, NO, and PGE2 were determined by enzyme-linked immunosorbent assay. Immunohistochemical analysis in the iris ciliary body (ICB) cells was perfomed to determine the expression of activated nuclear factor kappa B (NF-κB), inducible-nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2).Results: The oral administration with DSF suppressed, in a dose-dependent manner, the number of inflammatory cells, the protein concentration, and the levels of TNF-α, NO, and PGE2 in the aqueous humor and improved the histiologic status of the ocular tissue. The expression of activated NF-κB-positive cells in the ICB was significantly inhibited by oral administrated with DSF 3 hr after the LPS injection. The LPS-induced increased expressions of iNOS and COX-2 proteins in the ICB were also inhibited by oral DSF 24 hr after LPS injection.Conclusions: The present results indicate that oral DSF suppresses the inflammation in EIU by inhibiting the NF-κB-dependent pathway and the subsequent production of pro-inflammatory mediators.

The influence of inhibitors of α‐adrenergic receptors on uveitis induced by endotoxins

Abstract Purpose Investigation of anti‐inflammatory activity of Prazosin and Carvedilol in the experimentally induced anterior uveitis in rats. Activity of the Prazosin was compared to the activity of prostaglandin’s cyclooxygenase inhibitor – ibuprofen. Methods Prazosin suspended in PBS (phosphate buffered saline) was injected intra‐peritonealy (i.p.) in the dose of 2 to 30 mg/kg of body mass(b.m.) 24 and/or 3 hours prior to and/or 3hours after the induction of EIU – endotoxin induced uveitis – achieved by subcutaneous injection of 50 μg of the lipopolysaccharide (LPS) from Hafnia alvei 981. Carvedilol suspended in PBS was given i.p. in the dose of 30mg/kg b.m. 3hours before and after induction of EIU. Ibuprofen suspended in PBS was given i.p. in the dose of 100mg/kg b.m. 3hours before and after the EIU induction. Animals in the control group received PBS i.p. only. 24 hours following the EIU induction clinical assessment was performed with evaluation of the inflammatory cells in the anterior chamber fluid. Results Investigation of Prazosin’s influence on EIU induction showed, that when given 24hours prior to LPS injection it had no influence on inflammatory cells density or symptoms intensity. However, when given 3 hours before the induction, resulted in significant reduction of both parameters. Even stronger anti‐inflammatory reaction was observed, when given 3 hours post EIU induction. Doses reduced to 2 and 6 mg/kg b.m. proved to be active, when given in the same pattern. Administration of Prazosin 24 and 3 hours pre‐ and 3 hours post induction all together, blocked EIU induction. Conclusion Carvedilol proved to be far less active than Prazosin. It was also noticed that the Ibuprofen does not stop inflammation process.

Infectious and non infectious triggers in non‐infectious uveitis

Abstract Purpose The induction of autoimmune uveitis is difficult to explain with respect to the immune privileged status of the eye. The intact BRB can only be passed by already activated leukocytes, which should normally be ignorant to the sequestered intraocular antigens. Antigenic mimicry of retinal autoantigens by environmental proteins could explain extraocular activation of effector T cells. Methods We have previously demonstrated antigenic mimicry of a peptide from retinal S‐Antigen and peptides from rotavirus (Rota) and bovine milk casein (Cas). Both, Rota and Cas, induce T cell lines cross‐reactive with retinal S‐Ag peptide as well as experimental autoimmune uveitis in rats. Patients with uveitis have increased antibody and T cell responses to the mimicry peptides as well as to the S‐Ag peptide compared to healthy donors. Accordingly, Infection with rotavirus or any gastrointestinal pathogen with concomitant ingestion of bovine milk products could induce an immune response in the gastrointestinal tract that is cross‐reactive with ocular autoantigens and lead to induction of autoimmunity in the eye. Results Uveitis as a well known adverse effect after BCG (Bacille Calmette Guerin) treatment might also be the result of antigenic mimicry. We have shown T cell responses to PPD from M. tuberculosis and the retinal autoantigens S‐Ag, IRBP and CRALBP from a patient who had developed granulomatous uveitis after BCG application for bladder carcinoma. Data base searches revealed a number of amino acid sequence homologies between proteins from mycobacteria and retinal autoantigens, suggesting antigenic mimicry. These findings might as well be an explanation for the occurrence of uveitis in connection with M. tuberculosis infection, even when no mycobacteria are detectable in the eye.

Prevention of Endotoxin-Induced Uveitis in Rats by Plant Sterol Guggulsterone

To investigate the anti-inflammatory effects of guggulsterone, an antioxidant and antitumor agent, in endotoxin-induced uveitis (EIU) in rats and to elucidate the underlying molecular mechanism or mechanisms related to ocular inflammation. EIU was induced by subcutaneous injection of lipopolysaccharide (LPS; 150 μg) into Lewis rats treated with guggulsterone (30 mg/kg body weight, intraperitoneally) or its carrier. After 24 hours the rats were killed, eyes were enucleated, and aqueous humor (AqH) was collected. Numbers of infiltrating cells and levels of matrix metalloproteinase-2 (MMP-2), nitric oxide (NO), and prostaglandin E(2) (PGE(2)) were determined in AqH by specific ELISAs. An antibody array was used to measure the expression of various inflammatory cytokines in AqH. The expression of MMP-2, iNOS, Cox-2, phospho-IκB, and phospho-NF-κB was determined immunohistochemically. Human primary nonpigment ciliary epithelial cells (HNPECs) were used to determine the in vitro efficacy of guggulsterone on the LPS-induced inflammatory response. Compared with control, the EIU rat eye AqH had a significantly higher number of infiltrating cells, total protein, and inflammatory markers, such as MMP-2, NO, and PGE(2), and the treatment of guggulsterone prevented EIU-induced increases. Guggulsterone also prevented the expression of MMP-2, iNOS, and Cox-2 proteins and of IκB and NF-κB in various eye tissues. Moreover, in cultured HNPECs, guggulsterone inhibited LPS-induced expression of inflammatory proteins. These results for the first time demonstrate that the plant sterol guggulsterone suppresses ocular inflammation in EIU, suggesting that the supplementation of guggulsterone could be a novel approach for the treatment of ocular inflammation.

Treatment of experimental autoimmune uveitis in rats with arsenic trioxide

目的 探讨三氧化二砷(As2O3)对实验性自身免疫性葡萄膜炎(EAU)大鼠模型的治疗作用.方法 将光感受器间维生素A类结合蛋白(IRBP)R14多肽免疫大鼠随机分为高剂量As2O3组、低剂量As2O3组、环孢素A(CsA)组和生理盐水组4组,每组5只,分别腹腔注射5mg/kg As2O3、0.5mg/kg As2O3、2mg/kg CsA及生理盐水0.2mL.裂隙灯显微镜观察大鼠眼前房炎症情况,第14天时处死大鼠,光学显微镜观察大鼠眼球的病理变化程度,免疫组织化学观察大鼠眼球中CD3+ T细胞的浸润情况,TUNEL法检测大鼠视网膜组织的凋亡.结果 生理盐水组和0.5mg/kg As2O3组可见明显的虹膜充血、血管扩张和脓性渗出,5mg/kg As2O3组和CsA组前房炎症反应程度明显较轻,与生理盐水组比较,5mg/kg As2O3组和CsA组的临床评分明显降低,差异均有统计学意义(tAs2O3 5mg=-0.656,P=0.042;tCsA =-1.308,P=0.002).组织病理学评分显示各组间差异有统计学意义(F=11.297,P=0.000).与生理盐水组比较,As2O3组和CsA组的CD3+T细胞浸润明显减少.TUNEL检测细胞凋亡在各组间差异无统计学意义(P＞0.05).结论 腹腔注射As2O3可以明显抑制EAU大鼠的临床表现、病理改变及CD3+T细胞的浸润,未增加病变部位细胞的凋亡。

Anti-Inflammatory Effects of Lipoxins on Lipopolysaccharide-Induced Uveitis in Rats

Lipoxins exert potent anti-inflammatory and pro-resolving actions by reducing polymorphonuclear neutrophil (PMN) infiltration. This study describes the effect of lipoxin A4 and a stable analog on the resolution of ocular inflammation induced by intravitreal injection of lipopolysaccharides (LPS) in rats. Six- to eight-week-old male Sprague Dawley (SD) rats were injected intravitreally with 2.5 microL physiologically balanced solution (LPS) containing 5 ng LPS, or 5 ng LPS + 50 ng LXA4 or 5 ng LPS + 50 ng 15-epi-LXA4 analog. Rats were anesthetized with intraperitoneal injection of a ketamine and xylazine cocktail. At 24 h, the animals were again anesthetized and the eyes examined for clinical signs of inflammation. The animals were then euthanized by CO2 inhalation and aqueous humor was collected in heparinized saline. Aqueous humor PMNs were counted using an Improved Neubauer Hemocytometer, and the protein concentration was determined by standard procedure. After enucleation, the eyes were dissected to remove the lens and the ocular tissues were frozen in liquid nitrogen and stored at -80 degrees C. Myeloperoxidase assay was done by a standard procedure. Compared to untreated LPS-injected controls, rats treated with either LXA4 or its stable analog had lower clinical inflammation score, significantly reduced aqueous humor PMN cell counts, aqueous humor protein levels, and the MPO values. The difference between the mean values of aqueous humor protein and MPO in the LXA4 and the analog injected eyes was not statistically significant, but PMN cell counts were significantly different. The ocular inflammatory response to intravitreally injected LPS in rats is significantly reduced by simultaneous injection of LXA4 or its analog. This finding supports an earlier independent observation of the ocular anti-inflammatory effect of LXA4. Further investigation of lipoxins in the eye might offer a novel therapeutic approach to treating ocular inflammation in man.

Administration of Mycobacterium leprae rHsp65 Aggravates Experimental Autoimmune Uveitis in Mice

The 60kDa heat shock protein family, Hsp60, constitutes an abundant and highly conserved class of molecules that are highly expressed in chronic-inflammatory and autoimmune processes. Experimental autoimmune uveitis [EAU] is a T cell mediated intraocular inflammatory disease that resembles human uveitis. Mycobacterial and homologous Hsp60 peptides induces uveitis in rats, however their participation in aggravating the disease is poorly known. We here evaluate the effects of the Mycobacterium leprae Hsp65 in the development/progression of EAU and the autoimmune response against the eye through the induction of the endogenous disequilibrium by enhancing the entropy of the immunobiological system with the addition of homologous Hsp. B10.RIII mice were immunized subcutaneously with interphotoreceptor retinoid-binding protein [IRBP], followed by intraperitoneally inoculation of M. leprae recombinant Hsp65 [rHsp65]. We evaluated the proliferative response, cytokine production and the percentage of CD4+IL-17+, CD4+IFN-γ+ and CD4+Foxp3+ cells ex vivo, by flow cytometry. Disease severity was determined by eye histological examination and serum levels of anti-IRBP and anti-Hsp60/65 measured by ELISA. EAU scores increased in the Hsp65 group and were associated with an expansion of CD4+IFN-γ+ and CD4+IL-17+ T cells, corroborating with higher levels of IFN-γ. Our data indicate that rHsp65 is one of the managers with a significant impact over the immune response during autoimmunity, skewing it to a pathogenic state, promoting both Th1 and Th17 commitment. It seems comprehensible that the specificity and primary function of Hsp60 molecules can be considered as a potential pathogenic factor acting as a whistleblower announcing chronic-inflammatory diseases progression.

Arginase Activity Mediates Retinal Inflammation in Endotoxin-Induced Uveitis

Arginase has been reported to reduce nitric oxide bioavailability in cardiovascular disease. However, its specific role in retinopathy has not been studied. In this study, we assessed the role of arginase in a mouse model of endotoxin-induced uveitis induced by lipopolysaccharide (LPS) treatment. Measurement of arginase expression and activity in the retina revealed a significant increase in arginase activity that was associated with increases in both mRNA and protein levels of arginase (Arg)1 but not Arg2. Immunofluorescence and flow cytometry confirmed this increase in Arg1, which was localized to glia and microglia. Arg1 expression and activity were also increased in cultured Muller cells and microglia treated with LPS. To test whether arginase has a role in the development of retinal inflammation, experiments were performed in mice deficient in one copy of the Arg1 gene and both copies of the Arg2 gene or in mice treated with a selective arginase inhibitor. These studies showed that LPS-induced increases in inflammatory protein production, leukostasis, retinal damage, signs of anterior uveitis, and uncoupling of nitric oxide synthase were blocked by either knockdown or inhibition of arginase. Furthermore, the LPS-induced increase in Arg1 expression was abrogated by blocking NADPH oxidase. In conclusion, these studies suggest that LPS-induced retinal inflammation in endotoxin-induced uveitis is mediated by NADPH oxidase-dependent increases in arginase activity.

Prevention of Endotoxin-Induced Uveitis in Rats by Benfotiamine, a Lipophilic Analogue of Vitamin B1

To study the amelioration of ocular inflammation in endotoxin-induced uveitis (EIU) in rats by benfotiamine, a lipid-soluble analogue of thiamine. EIU in Lewis rats was induced by subcutaneous injection of lipopolysaccharide (LPS) followed by treatment with benfotiamine. The rats were killed 3 or 24 hours after LPS injection, eyes were enucleated, aqueous humor (AqH) was collected, and the number of infiltrating cells, protein concentration, and inflammatory marker levels were determined. Immunohistochemical analysis of eye sections was performed to determine the expression of inducible-nitric oxide synthase (iNOS), cyclooxygenase (Cox)-2, protein kinase C (PKC), and transcription factor NF-kappaB. Infiltrating leukocytes, protein concentrations, and inflammatory cytokines and chemokines were significantly elevated in the AqH of EIU rats compared with control rats, and benfotiamine treatment suppressed these increases. Similarly increased expression of inflammatory markers iNOS and Cox-2 in ciliary body and retinal wall was also significantly inhibited by benfotiamine. The increased phosphorylation of PKC and the activation of NF-kappaB in the ciliary body and in the retinal wall of EIU rat eyes were suppressed by benfotiamine. These results suggest that benfotiamine suppresses oxidative stress-induced NF-kappaB-dependent inflammatory signaling leading to uveitis. Therefore, benfotiamine could be used as a novel therapeutic agent for the treatment of ocular inflammation, especially uveitis.

Heat shock protein peptides reactive in patients with Behçet's disease are uveitogenic in Lewis rats.

Mycobacterial and homologous human heat shock protein T cell peptide epitopes specific for T lymphocytes in Behçet's disease were investigated for their pathogenicity in Lewis rats. The potential pathogenicity of eight peptides and two controls was assessed by administering the peptides in enriched Freund's adjuvant into the footpads of male Lewis rats. Anterior uveitis which is a major manifestation of Behçet's disease was induced with two out of the four mycobacterial and all four homologous human peptides. The most effective peptides inducing iridocyclitis in 64-75% of rats were peptides with amino acids 336-351 and 136-150, derived from the sequence of the human 60-kD heat shock protein. A few of the rats also showed evidence of focal loss of photoreceptors. These results suggest that selected peptides within heat shock protein 60 kD which function as T cell epitopes in Behçet's disease are capable of inducing uveitis in rats. This supports the view that the peptide T cell determinants may be involved in the pathogenesis of Behçet's disease.